Human Term Placental Mitochondria Research Articles

Glutamate in vitro effects on human term placental mitochondria

Objective: Oxidative stress may affect the functionality of placental mitochondria, thus contributing to serious complications. For this reason research of protective substances is of great importance. Our aim was to evaluate, in mitochondria isolated from human term placentas, the effect of in vitro glutamate supplementation on their susceptibility to oxidation, on the chemico-physical characteristics of mitochondrial membranes, and on peroxidase and nitric oxide synthase (NOS) activities. Methods: The study was performed on mitochondria isolated from 20 healthy human term placentas. Specific exclusion criteria were: conception by assisted reproduction, chromosomal or other fetal, uterine or placental anomalies, gestational diabetes, preeclampsia, intrauterine growth restriction (IUGR), a history of smoking and hypertension, proteinuria, renal, cardiovascular, hepatic, and endocrine disease, metabolic disorders, and current infection or history of all types of infection. Results: Incubation with glutamate determined a reduced susceptibility to oxidative stress, an increase in mitochondrial membrane fluidity, and a decrease of both peroxidase and NOS activities. Conclusions: On the basis of the observed results, we can hypothesize a role for glutamate in the control of lipid peroxidation extent in physiological pregnancies, as well as in the prevention of free radical-linked complications that can affect the health of both mother and fetus.

Differential effects of magnesium on the hydrolysis of ADP and ATP in human term placenta: Effect of substrates and potassium

This study evaluates the effect of Mg 2+ on the extramitochondrial hydrolysis of ATP and ADP by human term placental mitochondria (HPM) and submitochondrial particle (SMP). Extramitochondrial ATPase and ADPase activities were evaluated in the presence or absence of K +, and different oxidizable substrates. Mg 2+ increased both ATP and ADP hydrolysis according to the experimental conditions, and this stimulation was related to the mitochondrial intactness. The ADPase activity in intact mitochondria is 100-fold higher in presence of K +, succinate and 1 mM Mg 2+ while this activity is only increased by two-fold on the SMP when compared to the sample without Mg 2+. It is clearly demonstrated that up-regulation of these enzyme activities occur in intact mitochondria and not on the enzyme itself. The results suggest that the regulation of ATP and ADP hydrolysis is complex, and Mg 2+ plays an important role in the modulation of the extramitochondrial ATPase and ADPase activities in HPM

Presence of two enzymes, different from the F 1F 0-ATPase, hydrolyzing nucleotides in human term placental mitochondria

The hydrolysis of ATP, ADP or GTP was characterized in mitochondria and submitochondrial particles since a tightly-bound ATPase associated with the inner mitochondrial membrane from the human placenta has been described. Submitochondrial particles, which are basically inner membranes, were used to define the location of this enzyme. Mitochondria treated with trypsin and specific inhibitors were also used. The oxygen consumption stimulated by ATP or ADP was 100% inhibited in intact mitochondria by low concentrations of oligomycin (0.5 μg/mg) or venturicidine (0.1 μg/mg), while the hydrolysis of ATP or ADP was insensitive to higher concentrations of these inhibitors but it was inhibited by vanadate. Oligomycin or venturicidine showed a different inhibition pattern in intact mitochondria in relation to the hydrolysis of ATP, ADP or GTP. When submitochondrial particles were isolated from mitochondria incubated with oligomycin or venturicidine, no further inhibition of the nucleotide hydrolysis was observed, contrasting with the partial inhibition observed in the control. By incubating the placental mitochondria with trypsin, a large fraction of the hydrolysis of nucleotides was eliminated. In submitochondrial particles obtained from mitochondria treated with trypsin or trypsin plus oligomycin, the hydrolysis of ATP was 100% sensitive to oligomycin at low concentrations, resembling the oxygen consumption; however, this preparation still showed some ADP hydrolysis. Native gel electrophoresis showed two bands hydrolyzing ADP, suggesting at least two enzymes involved in the hydrolysis of nucleotides, besides the F 1F 0-ATPase. It is concluded that human placental mitochondria possesses ADPase and ATP-diphosphohydrolase activities (247).

Inhibitory Effect of Free Radicals Derived From Organic Hydroperoxide on Progesterone Synthesis in Human Term Placental Mitochondria

Different natural and synthetic organic hydroperoxides have been found to stimulate TBARS formation in human term placental mitochondria. The levels of TBARS were lower than arising from NADPH-dependent lipid peroxidation. BHT, Mn 2+ and DMPO counteracted TBARS formation in the presence of cumene hydroperoxide implicating involvement of free radicals in this process. On the other hand superoxide dismutase, catalase and EDTA while being inhibitory in NADPH-dependent lipid peroxidation did not inhibit cumene hydroperoxide-dependent TBARS formation. Amphenone B and SKF-525A, inhibitors of cytochrome P-450, strongly inhibit both NADPH- and cumene hydroperoxide-dependent lipid peroxidation. These data provide evidence that cytochrome P-450 SCC is involved in both these processes. However NADPH-dependent lipid peroxidation and the cumene hydroperoxide have been found to inactivate placental mitochondrial cytochrome P-450 SCC. The presence of cumene hydroperoxide resulted in a more rapid inactivation of cytochrome P-450 SCC and consequently inhibited NADPH-dependent lipid peroxidation. It has been observed for the first time that progesterone biosynthesis can be inhibited by cumene hydroperoxide. Protective effect of Mn 2+ and DMPO on progesterone biosynthesis indicates the importance of free radicals as transient products of cytochrome P-450 SCC-dependent cumene hydroperoxide metabolism. In contrast to progesterone formation from cholesterol, the conversion of pregnenolone to progesterone was not affected by cumene hydroperoxide. This suggests that inhibition of progesterone synthesis from cholesterol by hydroperoxide may be ascribed to its effect on the desmolase activity of cytochrome P-450 SCC in placental mitochondria. On the basis of the results obtained, we propose that the inhibition of progesterone biosynthesis by naturally occurring hydroperoxides may contribute to the development of preeclampsia.

Respiratory control induced by ATP in human term placental mitochondria

Oxygen uptake in human placental mitochondria was stimulated by ATP addition. ATP-induced respiration was supported by malate, alpha-keto glutarate, and succinate, and inhibited by oligomycin and carboxytractyloside. This phenomenon was not caused by contamination with unspecific phosphatases or alkaline phosphatase, since NaF, L-phenyl alanine, or P1, P5-di-(adenosine-5') pentaphosphate failed to inhibit oxygen uptake induced by ATP. The stimulation of respiration was caused by an ATPase activity tightly bound to mitochondria, which yields ADP that is responsible for the oxygen uptake. The stimulation was not an uncoupling effect because ATP addition produced a transition between state 3 and 4 of respiration, indicating that ATP was not released from mitochondria.

Phosphate-dependent glutaminase in the human term placental mitochondria

The properties of placental glutaminase described in this paper, namely relatively high substrate affinity, Hill coefficient about 1.6, inhibition by glutamate, and the lack of activation by bicarbonate make the placental enzyme very similar to the “kidney type” glutaminase isoenzyme of rat tissues.

Purification and properties of the NAD(P)-dependent malic enzyme from human placental mitochondria

The NAD(P)-dependent malic enzyme from human term placental mitochondria was purified 108-fold with a final yield of 72% and specific activity of about 2 μmol per minute per milligram protein. The final preparation was completely free of fumarase, malic, and lactic dehydrogenases. Divalent cations were required for NAD(P)-dependent malic enzyme activity, Mn 2+ and Co 2+ were by far more effective activators than Mg 2+ and Ni 2+, whereas the reaction did not proceed in the presence of Ca 2+. The optimum pH with NAD and NADP as coenzymes was at around 7.1 and 6.4, respectively. The ratio of the rate of NAD:NADP reduction was 7.4 and 1.3 at pH 7.1 and 6.4, respectively. The enzyme is activated by succinate and fumarate and inhibited by ATP. In the absence of fumarate the Michaelis constants for l-malate and NAD were 2.82 and 0.33 m m; and in the presence of fumarate 1.18 and 0.22 m m, respectively. This study presents the first report showing the purification and kinetic properties of NAD(P)-dependent malic enzyme from human tissue.

Correlation between the malate dependent progesterone and citrate biosynthesis in the mitochondrial fraction of human term placenta. The stimulatory effect of ADP and ATP

The relationship between malate dependent conversion of cholesterol to progesterone and citrate biosynthesis in human term placental mitochondria has been investigated. It has been shown that ADP and ATP (but not AMP) stimulate, significantly, both progesterone and citrate formation. The stimulatory effect of these adenine nucleotides was dependent on the presence of Mn 2+ in the incubation medium. When Mn 2+ was omitted or replaced by Mg 2+ only negligible stimulatory effect of ADP and ATP was observed. Atractyloside and oligomycin were without effect on ADP and ATP stimulated progesterone and citrate production. Other dinucleotides tested as: GDP, UDP and CDP stimulated both progesterone and citrate formation only slightly. In all the experiments presented the rate of progesterone biosynthesis was found to be significantly correlated with the rate of citrate production. The experimental results presented in this paper suggest that the stimulatory effect of ADP and ATP on malate dependent progesterone biosynthesis is a consequence of an increased conversion of malate to tricarboxylic Krebs cycle intermediates. The possible mechanism by which ATP and ADP stimulate the citrate formation in human placental mitochondria is discussed.

NAD(P)-dependent malic enzyme activity in human term placental mitochondria

Soluble protein obtained following centrifugation of sonicated mitochondria from human term placenta contained NAD(P)- and NADP-dependent malic enzyme activities. The NAD(P)-dependent activity catalyzing the oxidative decarboxylation of malate in the presence of either NAD or NADP (the former being much more effective) was localized almost exclusively in the fraction precipitating between 33 to 55% saturation of ammonium sulfate. The second, strictly NADP-dependent activity was present in the protein fraction precipitating between 55 to 70% saturation with ammonium sulfate. Mitochondria isolated from human term placenta and incubated in the presence of malate and Mn 2+ ions catalyzed pyruvate production that was stimulated by NADP or NAD. This suggests that the conversion of malate to pyruvate is catalyzed by NAD(P)-dependent malic enzyme.

Ammonia formation from some amino acids by human term placental mitochondria

The formation of ammonia from glutamine, glutamate, and branched-chain amino acids by human term placental mitochondria has been investigated. It has been shown that ammonia production from glutamine and glutamate was stimulated by NAD and ADP and was inhibited by oxoglutarate. Oxoglutarate substantially stimulated ammonia production from branched-chain amino acids. Ammonia production from branched-chain amino acids was inhibited by aminooxyacetate. The formation of lactate was observed during incubation of human term placental mitochondria with dicarboxylic Krebs-cycle intermediates and glutamate in the presence of lactate dehydrogenase. These results suggest that human term placental mitochondria are able to deaminate some amino acids and that the resulting carbon skeleton from glutamate could be converted to Krebs-cycle intermediates. The physiological significance of glutamate metabolism in human term placenta is discussed.

Partial characterization of placental 3ß-hydroxysteroid dehydrogenase (EC 1.1.1.145), Δ4–5isomerase (EC 5.3.3.1) in human term placental mitochondria

A partial characterization of human term placental 3ß-HSDH in mitochondria is reported. Apparent K M of pregnenolone: 70 nM. A dose-dependent stimulation of 3ß-HSDH by NAD + or NADP + was observed in the range from 10 −6 to 10 −3 M ( K M value of NAD +: 20 μM). At equimolar concentrations NAD + is more than 10-fold as effective a cofactor of the 3ß-HSDH than NADP +. pH optimum: 9.5 (glycine-NaOH buffer). Temperature optimum 40–45°C. A rapid loss of 3ß-HSDH activity was found after preincubation of the enzyme at 37°C after 30 min: less than 50% of initial enzyme activity is present. No inhibition was obtained by Mg 2+, Ca 2+ Sr 2+ and Ba 2+ (1–100 mM). A strong inhibition was achieved with 1 mM Zn 2+, Cd 2+, Cu 2+ and 10 mM and 100 mM Fe 2+, Mn 2+, Co 2+ and Ni 2+.

Oxidative phosphorylation reactions and cholesterol hydroxylation mechanisms in human term placental mitochondria

A simple and rapid method is described for the preparation of mitochondria of human term placenta. Phosphorylative oxidation properties were determined by polarography. Respiratory control values were higher than 3, showing that mitochondria are tightly coupled, but ADP/O ratios were lower than theoretical values due to ATPase activity. The activity of several specific mitochondrial marker enzymes was measured. In the outer membrane, rotenone-insensitive NADH-cytochrome c reductase and monoamine oxidase exhibited activities of 331 nmol of cytochrome c reduced/min/mg protein and 26.7 nmol of tyramine consumed/min/mg protein respectively. No kynurenine hydroxylase activity was detected. In the inner membrane, activities of succinate cytochrome c reductase and cytochrome oxidase were 15.3 nmol of cytochrome c reduced/min/mg protein and 21.6 nmol O 2 utilized/min/mg protein respectively. Glutamate, malate and succinate oxidases had low activities of about 1 to 3 nmol O 2 used/min/mg protein. In the matrix, glutamate dehydrogenase activity was very slight. Mitochondrial proteins were analyzed by polyacrylamide gel electrophoresis in the presence of phenol, urea and acetic arid. Mitochondria contained at least 20 components with mol. wt. ranging from 15,000 to 150,000. A component which probably contained cytochrome P450 subunits was characterized with a mol. wt. of 52,000. The concentration of the phosphorylating respiratory chain cytochromes was determined; the latter were in a similar range to that reported for adrenal cortex mitochondria, but their concentration was lower. The content of cytochrome P450 was lower than in other endocrine tissues. Oxygen consumption rate was stimulated by addition of (20α)-hydroxycholesterol and (22R)-hydroxycholesterol but not by (22S)-hydroxycholesterol. The placental cholesterol hydroxylating mechanism appeared to follow a pathway similar to that of adrenal cortex mitochondria.

Mitochondria from human term placenta: III. The role of respiration and energy generation in progesterone biosynthesis

The interrelationships among the processes of progesterone biosynthesis, respiration and energy production in human term placental mitochondria were examined. All substrates (citrate, isocitrate, α-ketoglutarate, succinate, fumarate, malate, glutamate, glycerol 3-phosphate, ascorbate + N, N, N', N'-tetramethyl-p-phenylenediamine) previously found to stimulate oxygen uptake and ATP synthesis in placental mitochondria supported progesterone synthesis from endogenous and exogenous cholesterol. Citrate support of progesterone synthesis was stimulated by NADP + but not NAD +. It was inhibited by fluorocitrate and trans-aconitate but not by arsenite, rotenone, antimycin, cyanide or 2,4-dinitrophenol. Ascorbate- N, N, N', N'-tetramethyl- p-phenylenediamine support of progesterone synthesis was stimulated by NAD + and NADP + and was inhibited by ADP, rotenone, antimycin, cyanide and 2,4-dinitrophenol. Cyanide inhibition was relieved by an exogenous ATP regenerating system and ADP inhibition was reversed by oligomycin. Progesterone synthesis supported by α-ketoglutarate + malonate was stimulated by NAD + and NADP +, and was completely inhibited by arsenite. 2,4-Dinitrophenol was strongly inhibitory both in the absence and presence of rotenone or antimycin. Stimulation by ATP was enhanced by rotenone, antimycin and oligomycin and inhibited by 2,4-dinitrophenol. Thus, metabolic control of progesterone synthesis by the energy status of the mitochondrial system was demonstrated when reducing equivalents were supplied via NADH or the respiratory electron transport chain.

Regulation of α-glycerophosphate dehydrogenase activity in human term placental mitochondria

1. 1. α-Glycerophosphate dehydrogenase ( sn-glycerol-3-phosphate:(acceptor) oxidoreductase, EC 1.1.99.5) activity in mitochondria isolated from human term placenta was found to be inhibited by ethyleneglycolbis(β-aminoethyl ether)-N,N′- tetraacetic acid (EGTA). Addition of an excess of calcium ions to the incubation medium completely restored the original activity. The concentration of free calcium ion required to activate the α-glycerophosphate dehydrogenase was found to vary between 10 and 100 nM. 2. 2. The pH optimum for α-glycerophosphate dehydrogenase activity varied with substrate concentration. The pH optima were 7.4 and 8.0 in the presence of 2 or 8 mM α-glycerophosphate, respectively. The apparent K m for α-glycerophosphate also varied with pH; the values being 0.4 mM at pH 7.05, 1.5 mM at pH 7.8, and 3.5 mM at pH 8.5. 3. 3. α-Glycerophosphate dehydrogenase activity was inhibited by palmitoylCoA in a competitive manner with an apparent K i value of about 10 μM. This inhibition was less pronounced in the presence of calcium or magnesium ions. 4. 4. The activity of α-glycerophosphate dehydrogenase was inhibited by phospho enolpyruvate, d- and dl-glyceraldehyde 3-phosphate and 3-phosphoglyceric acid, in a competitive manner, the apparent K i values being 0.5, 0.95, 0.12 and 1.5 mM, respectively. 5. 5. α-Glycerophosphate dehydrogenase activity in human placental mitochondria was found to be more sensitive to phospho enolpyruvate, than the activity of the same enzyme in rat skeletal muscle mitochondria, α-Glycerophosphate dehydrogenase activity in rat brown adipose tissue mitochondria was only slightly affected by phosph enolpyruvate under the same conditions. 6. 6. The data obtained suggest that the activity of α-glycerophosphate dehydrogenase in human placental mitochondria may be controlled by changes of the cytosolic level of palmitoyl-CoA, some glycolytic intermediates, and pH.

Oxidation of palmitoyl-carnitine by mitochondria isolated from human term placenta

It has been shown that slices prepared from either 13–14 week or term human placental tissue are able to oxidize palmitic acid as measured by collecting the evolved 14CO 2 from [1- 14C]palmitic acid (1,2). It has also been shown by Norum (3) that mitochondria isolated from this organ contain considerable amounts of carnitine palmitoyl transferase (EC 2.3.1.23) activity. This finding suggests that this enzyme plays a role in fatty acids oxidation by these mitochondria. To our knowledge fatty acids oxidation by mitochondria isolated from human term placenta has not yet been studied. This communication reports data indicating that isolated mitochondria from human term placenta oxidize palmitoyl-carnitine at the rate about 50% of the rate of isocitrate oxidation. Palmitic acid and palmitoyl-CoA alone were not oxidized under conditions examined. Palmitoyl-CoA in the presence of carnitine was oxidized at the rate about 50% of palmitoyl-carnitine oxidation. Palmitic acid in the presence of ATP, CoA, and carnitine was oxidized at the same extent as palmitoyl-CoA plus carnitine. The data obtained suggest that the formation of palmitoyl-carnitine is the rate limiting step in palmitoyl-CoA oxidation by human term placental mitochondria.

Immunochemical studies on adrenal ferredoxin: Involvement of adrenal ferredoxin-like iron-sulfur proteins in the cholesterol side-chain cleavage reaction of mammalian steroidogenic tissues

The possible immunochemical and functional similarities existing among adrenal ferrdoxin-like iron-sulfur proteins present in the mitochondria of mammalian steroidogenic tissues have been examined by employing a goat antibody produced against homogeneous bovine adrenal ferredoxin. This antibody was found to inhibit the NADPH-cytochrome c reductase and cholesterol side-chain cleavage activities catalyzed by mitochondria prepared from rat adrenals, rat ovaries and the testes of rats which had been treated with human chorionic gonadotropin. No inhibition of the NADH-dependent reduction of cytochrome c catalyzed by these mitochondria was observed in the presence of the anti-adrenal ferredoxin. These results demonstrate that adrenal ferredoxin and the comparable iron-sulfur proteins of ovarian and testicular mitochondria are immunochemically similar and are required for the cholesterol side-chain cleavage reaction occurring in these tissues. Although a precipitin reaction was observed upon double diffusion of the anti-adrenal ferredoxin against human term placental mitochondria, no inhibition of either the NADPH-cytochrome c reductase or the cholesterol side-chain cleavage 4 activity catalyzed by preparations of these mitochondria was observed in the presence of the antibody. These results indicate that the iron-sulfur protein present in human placental mitochondria at term differs immunochemically from other mammalian adrenal ferredoxin-like iron-sulfur proteins.

Mitochondria from human term placenta. I. Isolation and assay conditions for oxidative phosphorylation

Human term placental mitochondria were resolved by differential centrifugation into three fractions, heavy mitochondria, light mitochondria and a third, less dense fraction. Approximately equal amounts of mitochondrial protein were found in the three fractions. These mitochondrial preparations differed in physical properties, ATPase and “ADPase” content and oxidative capacities. Assay conditions were developed which permitted the polarographic measurement of respiration and coupled phosphorylation carried out by all three mitocondrial preparations despite the variable nucleotide-phosphate phosphatase activities present. With heavy mitochondria, rates of respiration were consistently higher than those previously reported for unfractionated placental mitochondria. Respiratory control ratios were comparable to those of mitochondria from other steroid hormone-producing endocrine tissues and ADP/O ratios approaching the theoretical maxima were obtained. Both lighter placental mitochondrial fractions displayed somewhat lower respiration rates and respiratory control but their primary defect was a selective uncoupling of the third site of energy conservation. Modification of isolation procedures were evaluated in terms of quantitative yield and functional activity of the three fractions.